Mosquitoes are vectors of numerous infectious diseases. To facilitate the development of advanced genome-based strategies for vector control, the genomes of the three major mosquito genera-Anopheles, Aedes, and Culex-were sequenced over the last decade. The genome assembly of Cx. quinquefasciatus, the major vector of the lymphatic filariasis worm and encephalitis viruses, is the most fragmented, with only 9% of the genome placed to the chromosomes. The lack of a high-quality, chromosome-based genome assembly for Cx. quinquefasciatus remains an impediment to further progress in Culex biology and comparative genomics of mosquitoes. Using the Next-Generation Focus Genome Sequencing (FGS) approach, recently developed by Amplicon Express, we will obtain ~250 Mb of high-quality sequence assemblies for 2304 BAC clones. Alignment of the sequences to the existing genome will greatly enhance the quality of the genome assembly. The total number of supercontigs will be reduced from 3171 to ~600. In addition, our project will develop a new cytogenetic map for Cx. quinquefasciatus based on mitotic chromosomes from imaginal discs of 4th instar larvae and anchor the majority of the genomic supercontigs to this map. The primary goal of this R21 proposal is to significantly improve the genome assembly for Cx. quinquefasciatus. The specific aims for this project are to: 1) improve the quality of the genome assembly by sequencing 2304 BAC clones using Focus Genome Sequencing (FGS) and 2) create a chromosome-based genome assembly by multicolor fluorescent in situ hybridization (FISH) of ~1000 BAC clones to Cx. quinquefasciatus chromosomes. The availability of a high-quality genome assembly for Cx. quinquefasciatus will enhance research in vector biology. Results of this project will be disseminated through VectorBase.